Traditionally, bacterial, yeast and mammalian cells are primarily cultured as suspension cultures in bioreactors, which are also, called fermenters. In such bioreactors the environmental conditions can be precisely controlled by manipulating the supply of nutrients to the cells and the removal of waste materials and a stirring means may move the culture medium in the interior of the reactor providing for a homogeneous distribution of the cells.
The bioreactor may be operated as a closed system such as a batch or fed-batch system or as a continuous system, such as a perfusion system or a chemostat system.
In a batch operation, the reactor is operated discontinuously. At the beginning of a batch, the culture medium usually contains a medium with the necessary nutrients, for example glucose, vitamins, amino acids and minerals. During fermentation, these are consumed so that the medium becomes more and more deprived in nutrients. At the same time, the concentration of waste products increases which finally results in a prevention of the cell growth. The viable cell density achieves a maximum value and thereafter decreases again. Consequently, the culturing is normally discontinued when the maximum cell density is reached or minimum cell viability is reached. The content of the reactor is then passed on for further downstream processing.
In this regard the so-called “feedbatch (alternatively named fed-batch) process” is a process in which, during the fermentation procedure, fresh culture medium is supplied (fed) to the bioreactor in order to supply more nutrients as those there are consumed during the bioreactor process. However, in practice this process has limitations to the volume that can be added and it does not provide for removal of waste products that might be harming the cell growth and productivity. Thus a feedbatch process does not provide any substantial advantages due to an increase of the waste materials.
The bioreactor may also be operated as a continuous system such as in a perfusion system or a chemostat system. In a perfusion system, the waste/impurities in the medium is continuously removed from the culture and the displaced medium is replenished with fresh medium. The constant addition of fresh medium and elimination of waste products provides the cells with the environment they require to achieve high cell concentrations and with that a higher productivity. Thus, it is possible to achieve a state of equilibrium in which cell concentration and productivity are maintained. Product may be continuously harvested by taking out medium (with cells and product).
A chemostat system is operated with a continuous inflow of medium and an outflow of cells and products keeping the culture volume constant. One of the most important features of chemostat systems is that microorganisms can be grown in a physiological steady state. In steady state, growth occurs at a constant rate and all culture parameters remain constant. Normally, in chemostat systems, there is no cell retention device, such that the concentration of cells in the bioreactor and the concentration of the cells in supernatant harvested from the bioreactor are substantially identical. Typically, culture medium is fed to the reactor at a predetermined and constant rate, maintaining a low dilution rate of the culture. To prevent washout of cells, the dilution rate generally is chosen to be less than, and sometimes equal to, the maximum specific growth rate of the cells. Culture fluids containing cells, cell products, by-products, waste products, etc., are removed through one common outlet at the same rate, or substantially the same rate. Compared to the perfusion system, the chemostat system typically results in lower cell densities and an inherent disadvantage of the chemostat systems is that the feed of the nutrients cannot be controlled independent of the product and/or cell harvest flow leading to a low productivity and high manufacturing cost of goods.
WO2011012725 describes a continuous cell culture strategy for producing polypeptides or viruses of interest in mammalian, cell culture wherein the cell culture system comprises a cell retention device consisting of a macroporous microcarrier such that the culture can be sustained for a prolonged period of time. However, WO2011012725 does not describe a method for producing a product in a chemostat fermentation process, wherein the bioreactor comprises one outlet having a separation device allowing impurities with a size below the size of the product, and medium to be removed and another independent outlet for removal of product, cells, cell products, by-products, waste products, etc.
In FIG. 1A herein is shown a typical prior art reactor having one inlet for adding medium and one outlet for harvesting the product of interest together with the culture fluids containing cells, cell products, by-products, waste products, etc. In other words, the continuous cell culture strategy described in e.g. WO2011012725 lacks the possibility to remove by-products, waste products and impurities through one outlet and harvest the product of interest through a second outlet.